Targeted Inactivation of DNA Photolyase Genes in Medaka Fish (Oryzias latipes)

• Published in: Photochemistry and photobiology Vol. 93; no. 1; pp. 315 - 322
• Main Authors: Ishikawa‐Fujiwara, Tomoko, Shiraishi, Eri, Fujikawa, Yoshihiro, Mori, Toshio, Tsujimura, Tohru, Todo, Takeshi
• Format: Journal Article
• Language: English
• Published: United States Blackwell Publishing Ltd 01.01.2017
• Subjects: Deoxyribonucleic acid; DNA; DNA repair; Genes; Light; Oryzias latipes; Ultraviolet radiation
• ISSN: 0031-8655; 1751-1097
• DOI: 10.1111/php.12658

Proteins of the cryptochrome/photolyase family (CPF) exhibit sequence and structural conservation, but their functions are divergent. Photolyase is a DNA repair enzyme that catalyzes the light‐dependent repair of ultraviolet (UV)‐induced photoproducts, whereas cryptochrome acts as a photoreceptor or circadian clock protein. Two types of DNA photolyase exist: CPD photolyase, which repairs cyclobutane pyrimidine dimers (CPDs), and 6‐4 photolyase, which repairs 6‐4 pyrimidine–pyrimidone photoproducts (6‐4PPs). Although the Cry‐DASH protein is classified as a cryptochrome, it also has light‐dependent DNA repair activity. To determine the significance of the three light‐dependent repair enzymes in recovering from solar UV‐induced DNA damage at the organismal level, we generated mutants in each gene in medaka using the CRISPR genome editing technique. The light‐dependent repair activity of the mutants was examined in vitro in cultured cells and in vivo in skin tissue. Light‐dependent repair of CPD was lost in the CPD photolyase‐deficient mutant, whereas weak repair activity against 6‐4PPs persisted in the 6‐4 photolyase‐deficient mutant. These results suggest the existence of a heretofore unknown 6‐4PP repair pathway and thus improve our understanding of the mechanisms of defense against solar UV in vertebrates. The harmful effects of UV radiation on organisms are reversed by concurrent or subsequent exposure to blue light. This is known as photoreactivation and is accomplished by DNA photolyase. We generated mutants for three photolyase genes, CPD photolyase, (6‐4)photolyase and cry‐dash, in medaka using the CRISPR genome editing method. The effect of gene knockout was most prominent in the CPD photolyase mutant, in which light‐dependent repair of CPD was lost. However, in the (6‐4) photolyase mutant, light‐dependent repair of 6‐4PPs was reduced dramatically, but some activity persisted. This unexpected result suggests the presence of an unknown light‐dependent repair mechanism.